The invention relates to methods for the acquisition of mass spectra in ion cyclotron resonance mass spectrometers, in particular to methods for exciting the ions to cyclotron trajectories. In ion cyclotron resonance mass spectrometers (ICR-MS), the charge-related masses m/z of the ions are determined by measuring their orbital frequencies in a homogeneous magnetic field with high field strength. The orbital motion is essentially a cyclotron motion on which a smaller magnetron motion is usually superimposed. The magnetic field is normally generated by superconducting magnet coils cooled with liquid helium. Nowadays, commercial mass spectrometers provide ICR measuring cells with usable diameters of up to approximately 6 centimeters at magnetic field strengths of between 7 and 15 tesla.
In ICR measuring cells, the orbital frequency of the ions is measured in the most homogeneous part of the magnetic field. The ICR cells normally comprise four longitudinal electrodes, which are parallel to the magnetic field lines and surround the interior of the measuring cell like a cylinder jacket. Cylindrical measuring cells are usually used, as shown in FIG. 1. The ions are introduced close to the axis. Normally, two opposing longitudinal electrodes, the “excitation electrodes”, are used to excite ions to their cyclotron motion by means of a pulse with alternating electric fields. Ions with the same charge-related mass m/z have to be excited as coherently as possible in order to achieve an in-phase orbiting cloud of these ions. The excitation to cyclotron motion brings the ions into circular orbits, whose diameter is usually around two thirds of the interior diameter of the ICR measuring cell. The two other electrodes, the “measuring electrodes”, serve to measure the orbiting of the ion clouds by image currents induced in the measuring electrodes as the ion clouds fly past.
The introduction of the ions into the measuring cell, ion excitation and ion detection are carried out in successive phases of the method. Various methods are available to introduce the ions into the ICR measuring cell and, in particular, for their capture, for example the “side-kick” method or the method of dynamic capture with a steady increase of the potential, but they will not be discussed further here. Those skilled in the art are familiar with these methods.
The ions are excited by absorbing energy in a dipolar alternating electric field between the two excitation electrodes. The frequency of the field must resonantly coincide with the cyclotron frequency of an ion species. The cyclotron frequency of the ions is inversely proportional to their mass m/z. Since the ratio m/z of the mass m to the number of elementary charges z of the ions (referred to below simply as “charge-related mass”, and sometimes simply as “mass”) is unknown before the measurement, the ions are excited by as homogeneous a mixture as possible of all the excitation frequencies for a desired mass range. This mixture can be a temporal mixture with frequencies linearly increasing or decreasing with time (called a “chirp”), or it can be a synchronous, computer-calculated mixture of all frequencies (a “sync pulse”). Commercial mass spectrometers usually operate with chirps; their initial and final frequencies, duration and voltage are chosen so that they lift ions of a selected mass range uniformly to a cyclotron trajectory with desired radius.
The ion image currents that are induced in the detection electrodes by the orbiting ion clouds form a co-called “transient” as a function of time. The transient is a “time-domain signal”. It usually starts with initially large ion image currents, which decrease during the measuring time to such a degree that only noise remains. The useful length of the transient up to the informationless noise is usually a few seconds, but in correctly adjusted ICR cells with compensation electrodes, as shown in FIG. 2, for example, it can last up to a few tens of seconds.
The ion image currents of the transients are amplified, digitized and analyzed by Fourier analysis to determine the orbital frequencies of the ion clouds occurring therein; the ion clouds each consist of ions of different masses orbiting in phase. The Fourier analysis transforms the sequence of the original ion image current values of the transient from the “time domain” into a sequence of frequency values in a “frequency domain”. ICR is therefore also called Fourier Transform Mass Spectrometry (FTMS), although it should be noted that, today, there are other types of FTMS which are not based on the orbiting of ions in magnetic fields.
The frequency signals of the various ion species, which can be recognized as peaks in the frequency domain, are then used to determine their charge-related masses m/z and their intensities. The high stability of the magnetic fields used and the high accuracy for frequency measurements make it possible to achieve an extraordinarily accurate mass determination. Fourier transform ICR mass spectrometers (FT-ICR-MS) are currently the most accurate of all types of mass spectrometer, with accuracies far better than one millionth of the mass for masses in the range up to around one thousand atomic mass units. FT-ICR-MS also provides the best mass resolutions, which are usually above one million for lighter ions, but which decline in inverse proportion as the mass of the ions increases. The mass resolution essentially depends on the number of ion orbits which can be detected by the measurement.
The transient usually looks like a very noisy signal which decreases roughly exponentially in time. The noise is only apparent; the signal very reproducibly consists of the superimposition of the many ion image current frequencies. FIG. 3 shows an example of a particularly long transient of the ion image currents of the doubly charged ions of “substance P”, which represents the typical shape of such a transient. The mass spectrum of the isotope group of these ions can be derived from this transient by Fourier transformation and further conversions, as is shown in FIGS. 4a and 4b. FIG. 4a shows the complete mass spectrum, which consists of the monoisotopic ions, the first 13C satellite and the second 13C satellite, and has a mass resolution of R=2,500,000. FIG. 4b shows the fine structure of the second 13C satellite in greatly enlarged detail; it is only observable with such a high mass resolution. Such measurements are useful in many ways; they can be used to quickly and easily determine the elementary composition of the substance under investigation, for example.
If, however, a particular mixture of ions consists of a larger number of ion species whose masses differ by the same mass difference in each case, the ion image current transient looks completely different. When the ions are excited by a standard chirp, so-called “beats” are formed in the transient when the image currents are measured. The ion clouds jointly lifted onto the cyclotron trajectory are initially all close together and produce the strong image currents of a first beat. The ion clouds of the slightly different masses, having slightly different speeds, then increasingly separate, however, and spread almost uniformly over the whole orbit over a long period; their image current signals appear to almost cancel each other out, as happens with an interference. Only when the ion clouds come close together again after many orbits is there a next “beat” of the image current. This process repeats periodically. The number of orbits nb between two beats is nb=M/ΔM, where M is the mass of the first ion of the group and ΔM is the mass difference between the different ions of the mixture.
These beats are especially common if one investigates organic substances with very high molecular weights. The ions of these substances are usually produced by electrospraying, which creates a broad distribution of multiply charged ions for large molecules. As an example, FIG. 5 shows a broadband mass spectrum of BSA (bovine serum albumin, molecular mass M=66,432.455 58 u). The signals of the protonated molecular ions with 32 to 63 charges can be seen. For substances with very high mass in the order of several ten thousands of atomic mass units, commonly at first a broadband overview spectrum is acquired, and then a narrowband mass spectrum showing only the ions of one charge state at maximum resolution. These mass spectra with very high mass resolution are analytically very useful; they can be used to identify not only the elemental composition, but also derivatization states, the purity of the substance and associations with smaller molecules.
With heavy organic substances, the ions of one charge state form an isotope group with often far more than a hundred isotope satellites. Since the ions of this isotope group each differ by one atomic mass unit (to be more precise, by the mass difference between 12C and 13C in each case), they constitute a very uniformly structured ion mixture, which forms a transient with pronounced beats on being excited with a chirp, as can be seen in FIG. 6 for the protonated molecular ions of BSA with 49 charges.
The information contained in the transients is not only found in the beats, but also in the spaces between the beats, which visually appear to be almost empty. In these spaces, the image current frequencies are superimposed in a similar way to the “normal” transient of FIG. 3. In order to measure the image current values in the spaces efficiently, the dynamic measurement range must be extraordinarily large. The usually already high dynamic measurement range of 20 bits in commercial ICR electronics is not sufficient for this. Special measures are required to obtain the full information that is contained in the measured values of such a transient with strong beat. The special measures usually consist in acquiring the image current transient not only once, but many hundreds of times.
The mass spectrum of the isotope signals of the BSA ions with 49 charges, which is shown in FIGS. 7a, 7b and 7c in three magnifications, could only be measured well, and even with a mass resolution of R=800,000, because the transients of 200 image current measurements have been summed. Since each transient had a length of 15 seconds, the complete measuring time here was 3,000 seconds or 50 minutes. Such a long measuring time is not acceptable for many analytical tasks. Moreover, a successful summation of 200 individual spectra demands not only a stable magnetic field, but also an extraordinarily high stability of all the electrical parameters in the electronics, which is rarely the case.
As stated above, the beats are produced by an interference behavior of the ions during their orbits. The excitation lifts the ions to a cyclotron trajectory where all the ion clouds are initially very close together and result in a strong ion image current signal, the first beat. Then the ion clouds, which each differ by a tiny fraction of their relative mass and thus by a tiny fraction of their speed, slowly drift apart and distribute themselves almost evenly over the complete cyclotron orbit. When the distribution is even, however, the ion image current signals almost cancel each other out; the intensity of the signals is very low and can hardly be measured next to the intense beats. In the case of BSA, all the ions then come together again after 66,389 orbits of the monoisotopic ions; the ions of the first 13C satellite mass pass through one orbit less than the monoisotopic ions, the ions of the second satellite two fewer orbits, the ions of the third satellite three fewer orbits, etc. This produces the second beat. The ion species then spread out again until they meet up once again after a further 66,389 orbits to form a third beat.
This process continues periodically but the beats become smaller and smaller because, although the mass differences are identical over the whole mixture, this is not the case for the differences of the speeds, which are reciprocal to the masses, as can easily be mathematically verified. Since the differences in the speeds are only equal in the first approximation, albeit a very good approximation, the ions meet up less and less after each successive 66,389 orbits, and their beat becomes smaller.
The chirps used in the current prior art have a linear frequency function with the same amplitude for all frequencies, as is shown in FIG. 8. In commercial ICR mass spectrometers, the initial frequency, final frequency, duration and amplitude (voltage) of the chirp are usually adjustable. The frequency range is from a few kilohertz up to around 100 kilohertz; the voltage can be set between a few volts and around 300 volts; the duration of the chirp can be up to 20 milliseconds or more.
To describe the effect of a chirp, we will now assume it to be a chirp with linearly increasing frequency. For this chirp with linearly increasing frequency function, the ions are excited in a sequence from heavy to light masses. If the increasing frequencies of such a linear chirp cover the cyclotron resonance frequencies of all the ions of an ion mixture in roughly ten milliseconds, the lightest ions reach their cyclotron orbit some ten milliseconds later than the heaviest ions. The temporal separations of the ions for reaching the orbit are proportional to their mass difference.
If the ion mixture consists of ions with the same mass differences throughout, all ions catch up with the heaviest ions simultaneously because the light ions fly slightly faster on their orbit and because the temporal separations between the lighter ions and the heavier ones are inversely proportional to their speed on reaching the orbit. All the light and heavy ions will therefore come together at the same time, resulting in the first beat.
The image currents are measured using amplifiers which offer a wide range of amplification adjustment, and analog-to-digital converters (ADC) with 16 to 20 bit conversion width. The latter determine the dynamic measuring range with which a transient can be measured.
Where the term “acquisition of an ICR mass spectrum” or similar wording is used below, this encompasses the complete sequence of steps: filling the ICR measuring cell with ions, exciting the ions to cyclotron trajectories, measuring the image current transient, digitizing, Fourier transformation, determining the frequencies of the individual ion species and, finally, calculating the charge-related masses m/z and intensities of the ion species which constitute the mass spectrum, as is known by anyone skilled in the art.